Searching for New Antimalarial Therapeutics amongst Known Drugs

Abstract

The need to discover and develop new antimalarial therapeutics is severe. The annual mortality attributed to malaria, currently approximately 2.5 million, is increasing due primarily to widespread resistance to currently used drugs. One strategy to identify new treatment alternatives for malaria is to examine libraries of diverse compounds for the possible identification of novel scaffolds. Beginning with libraries of drug or drug-like compounds is an ideal starting point because, in the case of approved drugs, substantial pharmacokinetic and toxicologic data should be available for each compound series. We have employed a high throughput screen of the MicroSource Spectrum and Killer collections, a library of known drugs, bioactive compounds, and natural products. Our screening assay identifies compounds that inhibit growth of Plasmodium falciparum cultured in human erythrocytes. We have identified 36 novel inhibitors of P. falciparum, of which 19 are therapeutics, and five of these drugs exhibit effective 50% inhibitory concentrations within similar ranges to therapeutic serum concentrations for their currently indicated uses: propafenone, thioridazine, chlorprothixene, perhexiline and azlocillin. The findings we report here indicate that this is an effective strategy to identify novel scaffolds and therefore aid in antimalarial drug discovery efforts.

Assessment and Continued Validation of the Malaria SYBR Green I-Based Fluorescence Assay for Use in Malaria Drug Screening

“…We found the assay quality to be more than fit for high-throughput screening efforts (0.73 Ն ZЈ Յ 0.95), regardless of the culture conditions or parasite strain used. Our value range is similar to that previously described for YOYO-1 (median ZЈ of 0.75) (37) and DAPI (ZЈ ϭ 0.799) (3). In addition, the assay appears to work very well in both 384-and 1536-well formats (Elizabeth Winzeler, unpublished data).…”

“…The quality or robustness of the assay for high-throughput screening was calculated by using the statistical measure of ZЈ (40) as described in Materials and Methods. Assays that display a ZЈ value of Ն0.5 are generally acceptable for highthroughput screening (HTS) (37). To determine this value, half a plate each of noninfected and D6-infected erythrocytes (starting at late-ring or early-trophozoite stages) in the absence of antimalarial drugs were cultured for 72 h and analyzed by using the MSF assay.…”

“…Several in vitro antimalarial drug surveillance microtiter plate assays that involve sensitive nonradioactive procedures have been recently published (3,7,10,28,33,37). For example, a few laboratories have described colorimetric enzyme-linked immunosorbent assays (ELISAs) that recognize specific P. falciparum antigens (10,28).…”

“…Also, Corbett et al showed the utility of the fluorescence-based assay for the screening of novel antimalarial compounds derived from crude plant extracts in the same report. YOYO-1 was used to search known drugs and bioactive compounds for new antimalarials, resulting in the identification of several novel P. falciparum inhibitors (37). Most recently, Banieki et al demonstrated the robustness and compatibility of the fluorescent dye DAPI (4Ј,6Ј-diamidino-2-phenylindole) for monitoring Plasmodium growth in high-throughput antimalarial drug screening using a 384-well microtiter plate (3).…”

“…In addition to ELISAs, assays involving fluorescent nucleic acid intercalating dyes, such as SYBR green I, PicoGreen, and YOYO-1, to measure in vitro malaria growth inhibition have been recently described (3,4,7,16,31,33,37). Since mature erythrocytes lack RNA and DNA, binding of the dye is specific for malarial DNA in any erythrocytic stage of P. falciparum development (4).…”